synthesis and conjugation of neisseria meningitidis x

Issue in Honor of Prof Richard R. Schmidt ARKIVOC 2013 (ii) 166-184

66 ©ARKAT-USA, Inc.

Synthesis of Neisseria meningitidis X capsular polysaccharide

fragments

Laura Morelli and Luigi Lay*

Dipartimento di Chimica and ISTM-CNR, Università degli Studi di Milano,

via Golgi, 19 I-20133 Milano, Italy

E-mail: [email protected]

Dedicated to Professor Richard R. Schmidt on the occasion of his 78th anniversary

DOI: http://dx.doi.org/10.3998/ark.5550190.0014.214

Abstract

Serotype X of Neisseria meningitidis bacterium (Men X) recently emerged as a substantial threat

to public health. Since anti-meningococcal vaccines currently available or under investigation do

not contain antigenic components of Men X capsular polysaccharide, there is the need to develop

more comprehensive conjugate vaccines capable to offer higher protection. As a preliminary step

towards this goal, the synthesis of three conjugatable Men X capsular polysaccharide fragments

is described. The installation of the crucial α-glycosyl phosphodiester linkages is based on the

hydrogenphosphonate methodology using pure α-glycosyl hydrogenphosphonates 10 and 12

obtained from hemiacetals 9 and 11, respectively.

Keywords: Carbohydrates, glycosyl phosphates, hydrogenphosphonates, Neisseria meningitidis

Introduction

Bacterial meningitis causes approximately 170,000 annual deaths upon more than 1,200,000

cases, with at least a 5-10% of case fatality in industrialized countries and a 20% in the

developing world.1 Streptococcus pneumoniae, Haemophilus influenzae type b (Hib) and

Neisseria meningitidis are responsible for most of the cases of bacterial meningitis worldwide.2

In particular, thirteen different serogroups of N. meningitidis have so far been defined, but about

90% of the infections are due to serogroups A, B, C, Y and W135.3 However, in the past 20

years sporadic cases or clusters of meningitis due to other N. meningitidis serogroups have

emerged. N. meningitidis type X (Men X), first described in the 1960s,4 has been found to cause

a few cases of invasive disease across North America, Europe, Australia and Africa.5 In 2006,

owing to an unprecedented incidence of meningitis caused by Men X in Niger,6 the World

Health Organization (WHO) recognized Men X as a substantial threat and epidemic potential.

mailto:[email protected]

http://dx.doi.org/10.3998/ark.5550190.0014.214


Page 167 ©ARKAT-USA, Inc.

Vaccination is considered by the WHO to be the most cost-effective strategy for controlling

infectious disease, since it should confer long-term protective immunity in the population. In

particular, carbohydrate-based vaccines have recently emerged as a powerful tool with enormous

potential benefits for human health.7 They are designed to target pathogen-specific cell surface

saccharide structures, such as the carbohydrate capsule (capsular polysaccharide, CPS), which

represent a major virulence factor of encapsulated bacteria, including N. meningitidis. Saccharide

vaccines currently present on the market, or under development, are based on carbohydrate-

protein conjugates,8 and they comprise also different formulations against H. influenzae type b,

S. pneumoniae and several meningococcal serogroups. However, none of them include antigenic

components of Men X, and therefore they do not offer protection against infections caused by

this emerging serogroup. Although Men X currently causes only a small proportion of

meningococcal disease,5,6 it is possible that repeated vaccination against some serogroups

(especially A and C) has the potential to select meningococci of other serogroups (for example,

Men X) and might result in a changed profile of meningococcal disease. This possibility should

be considered when conjugate vaccines carrying limited ranges of serogroups are introduced.5b

The development of more comprehensive conjugate vaccines including Men X CPS fragments

could therefore become an urgent issue in the near future.

Carbohydrate-based antigens needed for inclusion in a vaccine, however, are not readily

available from natural sources. The isolation and purification of the polysaccharide from the

bacterial source is indeed a challenging task, leading to the presence of biological contaminants

and impurities, thus raising severe issues of quality assurance. On the other hand, synthetic

carbohydrate-based vaccines have important advantages, including their well-defined chemical

structures and a better safety profile. Consequently, a variety of pathogen-associated saccharide

antigens have been synthesized in the last decade, coupled to carrier proteins and proved to elicit

protective antibodies in animal models.9 In addition, synthetic oligosaccharides can help

elucidate the structural moieties of the native bacterial polysaccharide that are essential to induce

antibodies.10 This step is crucial for the design of a new generation of improved and safer

vaccines obtained either from chemical synthesis or bacterial source.

On the basis of these considerations, we report herein the synthesis of the monomer, dimer

and trimer of Men X CPS repeating unit (compounds 1-3, Figure 1). The synthetic fragments are

provided with a phosphodiester-linked aminopropyl spacer at their reducing end, suitable for

their eventual conjugation to a carrier protein.

Figure 1. Structures of repeating unit of Men X CPS and target oligomers 1-3.



Results and Discussion

The CPS of N. meningitides X is a homopolymer of (14)-linked 2-acetamido-2-deoxy- α-D-

glucopyranosyl phosphate residues (Figure 1).11 There is no doubt that the major challenge in the

preparation of our target oligomers is the stereochemical control in the synthesis of the α-

phosphodiester linkages. Among other methodologies available in the literature for the formation

of phosphodiester linkages, we relied on the well-established hydrogenphosphonate (H-

phosphonate) protocol.12 The same strategy was employed by Shibaev and co-workers who, to

the best of our knowledge, reported the first synthesis of Men X dimer.13 The Russian group

synthesized an anomerically pure α-H-phosphonate (glycosyl phosphite) of a N-

acetylglucosamine (GlcNAc) derivative which was then coupled with the 4-OH of the O-p-

nitrophenylglycoside of a second GlcNAc residue. On the other hand, our fragments are intended

for the preparation of the corresponding protein conjugates, and each compound contains a

phosphodiester-linked spacer available for this purpose. An additional advantage of our approach

is the higher flexibility, since the protecting groups pattern of the synthetic precursors was

designed to allow further elongation and synthesis of oligomers with variable length, as

demonstrated by the preparation of trimer 3. Accordingly, suitably protected azide-containing

monosaccharides 4 and 5 (Scheme 1) were employed as key building-blocks. In particular,

compound 5 was selected as elongation block, since the 4-OH is easily available by Zemplén

transesterification, whereas we envisaged intermediate 4 as a capping block to be introduced at

the non-reducing terminus of each oligomer. Compound 4 -precursor of 5- was initially

synthesized in four linear steps from D-glucosamine hydrochloride as described by Martin-

Lomas et al.14 This procedure resulted however unsuitable for large laboratory scale (over 20 g

of starting material), and we applied the longer but more reliable reactions sequence outlined in

Scheme 1, which provided intermediates 4 and 5 in good overall yield and much higher purity.

Briefly, D-glucosamine was first converted into the 2-azidoglucose derivative 7 by diazotransfer

reaction followed by standard O-acetylation (Ac2O, pyridine, N,N-dimethylaminopyridine,

DMAP) in 77% yield. The crucial diazotransfer reaction was carried out using the recently

described15 imidazole-1-sulfonyl azide diazo donor as a cheap and safe alternative to the more

popular trifluoromethanesulfonyl azide (TfN3).16 Besides the high cost of

trifluoromethanesulfonic anhydride, used in its preparation, neat TfN3 has indeed explosive

nature and its poor shelf life requires its preparation in solution just prior to use.17 On the

contrary, the imidazole-1-sulfonyl azide can be easily prepared from relatively inexpensive

starting materials and its hydrochloride salt (compound 6 in Scheme 1) is a crystalline solid that

can be stored at 4°C for weeks without loss of efficiency.



Scheme 1. Synthesis of key building-blocks 4 and 5.

Regioselective 1-O-deacetylation followed by 1-O-silylation with thexyldimethylsilyl

chloride (TDSCl) and imidazole in DMF afforded intermediate 8 in good yield (66% yield over

two steps). This compound was converted into building-block 4 by Zemplén O-deacetylation,

4,6-O-benzylidenation and final 3-O-benzylation (45% yield over three steps). Eventually,

regioselective reductive opening of the benzylidene acetal in 4 (Et3SiH and trifluoroacetic acid),

followed by standard 4-O-acetylation provided monosaccharide 5 in high yield (78% yield over

two steps).

Next stage of the synthesis of the target oligomers was the crucial installation of the

anomeric phosphodiester linkages using the H-phosphonate protocol. We reasoned that the most

reliable way to obtain the stereochemical control in the synthesis of anomerically pure α-glycosyl

phosphodiesters is the condensation of a glycosyl α-H-phosphonate with the free hydroxyl of a

sugar acceptor, followed by oxidation of the H-phosphonate diester intermediate. The

preparation of anomerically pure glycosyl α-H-phosphonate is however a challenging task.

Accordingly, no examples have been found in the literature describing the synthesis of

exclusively α-anomeric H-phosphonate from 2-azido-2-deoxy-glucopyranosyl derivatives.

Literature data suggest however that the treatment of a sugar hemiacetal with a proper

phosphitylating agent in the presence of phosphorous acid could lead to equilibration of the

initially formed α,β mixture of the anomeric H-phosphonates into the pure, more

thermodynamically stable α anomer.18 Compound 4 was therefore 1-O-desilylated using

tetrabutylammonium fluoride and acetic acid in THF at -40°C to afford hemiacetal 9 in 80%

yield (Scheme 2). The synthesis of the α-H-phosphonate from 9 was next thoroughly

investigated testing different reaction conditions, varying the phosphitylating agent, the reaction

solvent and temperature, and the stoichiometry of the reagents. Eventually, we found that the

treatment of 9 with 2-chloro-4H-1,3,2-benzodioxaphosphinin-4-one (commonly named

salicylchlorophosphite) and H3PO3 in 1:1.5:3 molar ratio in pyridine from room temperature to

40°C (see the Experimental Section) provided exclusively α-H-phosphonate 10, as evinced by 1H- and 31P-NMR spectra. The formation of the H-phosphonate was confirmed by the diagnostic



doublet at 7.01 ppm in the 1H-NMR spectrum with the characteristic value of 1JH,P 640.4 Hz (δP

1.84), while the α-configuration was ascertained by the signal corresponding to H-1 (δH 5.70, J1,2

3.5 Hz, J1,P 8.8 Hz).

Scheme 2. Synthesis of α-H-phosphonates 10 and 12.

The long reaction time (8-9 days are needed) represents a significant drawback of this procedure.

The reaction progress could be however easily monitored by 1H- and 31P-NMR analysis. As

shown in Figure 2 (part a), as the reaction proceeds one can observe the progressive

disappearance of the signals corresponding to the β-glycosyl H-phosphonate with the

concomitant formation of the hemiacetal 9 (see the doublet at δ 5.32). Figure 2b shows the same

trend in 31P-NMR spectra. According to the literature,18 a reasonable explanation of the observed

behaviour is that the more reactive β-glycosyl H-phosphonate is converted to either the α-anomer

10 (due to a SN2 displacement by H3PO3) or the hemiacetal 9 as a result of acid-catalyzed

cleavage of the H-phosphonate group. In accordance with this mechanism, the α-H-phosphonate

10 was obtained in 41% yield along with 45% of the hemiacetal 9, which could be easily

separated by chromatography and recycled.

Figure 2. 1H-NMR (part a) and 31P-NMR spectra (part b) showing the reaction progress from 9

to 10.



The same protocol was successfully applied to hemiacetal 11, obtained by 1-O-desilylation

of compound 5, affording the α-glycosyl H-phosphonate 12 in 52% yield (along with 38% of

recovered 11, Scheme 2). The formation of α-H-phosphonate 12 from 11 was slightly faster (6-7

days), and its structure was confirmed by 31P- and 1H-NMR data (δP 2.24, δH 7.03 for H-P with 1JH,P 642.0 Hz, δH 5.73 for H-1, with J1,2 3.0 Hz and J1,P 8.7 Hz). NMR spectra of the reaction

mixture during the conversion of 11 into 12 are illustrated in Figure 3.

Figure 3. 1H-NMR (part a) and 31P-NMR spectra (part b) showing the reaction progress from 11

to 12.

Now the stage was set for the assembly of the target oligomers. First, glycosyl H-

phosphonate 10 was coupled with commercially available benzyl N-(3-hydroxypropyl)carbamate

using pivaloyl chloride as condensing agent (Scheme 3).12,19 In situ oxidation of the H-

phosphonate diester intermediate was carried out with iodine in aqueous pyridine, providing

phosphodiester 13 in 62% yield as triethylammonium salt after quenching the reaction mixture

with 1 M aq. triethylammonium hydrogen carbonate (TEAB) buffer solution (pH 7). The

structure of compound 13 was confirmed by NMR and mass spectrometry. The 31P-NMR

spectrum exhibited a single signal with δP -0.84 characteristic for glycoside-linked

phosphodiesters.

The subsequent azide reduction turned out much more difficult than expected. While the

classical Staudinger reaction20 failed, all other attempts (1,3-propanethiol, PMe3, Zn-Cu couple

in THF-Ac2O-AcOH) gave disappointing results, with only traces of the desired product. Finally,

the azide was converted into the acetamide using a combination of NiCl2 and NaBH4 followed

by treatment with Ac2O,21 affording compound 14 in 29% yield. In spite of this unsatisfactory

result, we deemed this yield acceptable at this stage of the research, and the obtained acetamido



intermediate 14 was submitted to final deprotection. The remaining protecting groups were

removed via hydrogenolysis over Pd on carbon. Final purification was accomplished by eluting a

water solution of deprotected compound over a column filled with Dowex 50W X8 resin (H+

form), followed by a second ion exchange on the same resin in Na+ form. Lyophilization of the

eluates provided the spacer-linked monomer 1 as sodium salt in 93% yield (Scheme 3).

Scheme 3. Synthesis of the spacer-linked monomer 1.

The syntheses of dimer and trimer required the condensation of the glycosyl H-phosphonate

12 with benzyl N-(3-hydroxypropyl)carbamate in the presence of pivaloyl chloride, followed by

in situ oxidation to phosphodiester 15 (64%) and careful Zemplén 4-O-deacetylation (96% yield,

Scheme 4). The resulting alcohol 16 was coupled with the capping residue 10 to afford, after

oxidation, phosphodisaccharide 17 in 45% yield. The azide reduction was accomplished using

the NiCl2/NaBH4 protocol, followed by N-acetylation and hydrogenolytical removal of the

protecting groups, furnishing after ion exchange fully deprotected dimer 2 as bis-sodium salt in

36% overall yield (from 17, Scheme 4).

On the other hand, the synthesis of trimer 3 was achieved by condensation of alcohol 16 with

the elongation block 12 followed by oxidation, which gave phosphodisaccharide 18 in 40% yield

(Scheme 5).



Scheme 4. Synthesis of the spacer-linked dimer 2.

Scheme 5. Synthesis of the spacer-linked trimer 3.



Zemplén deacetylation of 18 and subsequent coupling with 10 provided phosphotrisaccharide

20 in 43% overall yield after oxidation. Reduction of the three azides and N-acetylation was

carried out with NiCl2/NaBH4 followed by Ac2O addition. Eventually, hydrogenolysis of the

remaining protecting groups and ion exchange performed as described above afforded trimer 3 as

tris-sodium salt in 33% overall yield from 20 (Scheme 5).

Oligomers 2 and 3 were fully characterized by NMR spectroscopy and mass spectrometry. 1H- and 13C-NMR spectra, using both mono- and bidimensional experiments, were in excellent

agreement with the proposed structures (see the Experimental Section). In addition, 31P-NMR

spectra showed two peaks at δ 0.90 and 0.60 for dimer 2, and three peaks (δ -0.31, -0.65 and -

0.71) for trimer 3.

Conclusions

In conclusion, as a preliminary step towards the development of more comprehensive anti-

meningococcal conjugate vaccines including new emerging serotypes, we described the

synthesis of three conjugatable fragments (monomer 1, dimer 2 and trimer 3) with structures

corresponding to the natural CPS of N. meningitidis type X. The challenging synthesis of

anomerically pure α-hydrogenphosphonate, a crucial aspect for our strategy, has been

accomplished for the first time on 2-azido-2-deoxy derivatives using a combination of H3PO3

and salicylchlorophosphite. Our approach is featured by high flexibility and, in principle, it

allows the synthesis of even longer oligomers by iteration of the 4-O-deacetylation-

hydrogenphosphonate condensation sequence either using the capping residue 10 or the

elongation block 12. On the other hand, a major drawback of our approach are the disappointing

results obtained in the reduction of the azido functions, which caused a significant drop of the

chemical yields. The use of the azido group was due to its non-participating and electron-

withdrawing properties, that were expected to enhance the stability of the anomeric

phosphodiester linkages. The difficulties encountered in the final deprotection of the oligomers

however induced us to take into account a different approach, based on GlcNAc instead of azido

glucose building blocks, which is currently under investigation.

The chemical conjugation of oligomers 1-3 to immunogenic carrier proteins, and the

biological evaluation of the resulting neo-glycoconjugates will be reported in due course.

Experimental Section

General. All commercially available reagents including dry solvents were used as received. The

only exception is H3PO3, which was coevaporated three times with dry toluene, and dried by

high vacuum pump prior to use. Reactions were monitored by thin-layer chromatography on pre-

coated Merck silica gel 60 F254 plates and visualized by staining with a solution of cerium



sulfate (1 g) and ammonium heptamolybdate tetrahydrate (27 g) in water (469 mL) and

concentrated sulfuric acid (31 mL). Chromatographic purifications have been performed either

using the flash purification apparatus Biotage® SP1™ by gradient elution, or by standard

column chromatography on Fluka silica gel 60. NMR spectra were recorded at 300K on

spectrometer operating at 400 MHz. Proton chemical shifts are reported in ppm (δ) with the

solvent reference relative to tetramethylsilane (TMS) employed as the internal standard (CDCl3 δ

7.26 ppm). J values are given in Hz. Carbon chemical shifts are reported in ppm (δ) relative to

TMS with the respective solvent resonance as the internal standard (CDCl3, δ 77.0 ppm). In 13C-

NMR spectra, signals corresponding to aromatic carbons are omitted and, apart quaternary

carbons, signals attribution was derived by HSQC experiment. Signals attribution in NMR

spectra are designated using indexes a, b and c, and referred to ring a (reducing end, directly

linked to the spacer), ring b (either the non-reducing end or the inner ring), and ring c (the non-

reducing terminus). Optical rotations values are given in 10-1 deg cm2 g-1 and were measured at

25 °C on a polarimeter at 589 nm using a 5 mL cell with a length of 1 dm. High resolution mass

spectra (HRMS) were performed at CIGA (Centro Interdipartimentale Grandi Apparecchiature),

with mass spectrometer APEX II & Xmass software (Bruker Daltonics). ESI-MS spectra were

recorded on a JEOL AX-505 spectrometer.

1,3,4,6-Tetra-O-acetyl-2-azido-2-deoxy-D-glucopyranose (7). Imidazole-1-sulfonyl azide

hydrochloride 615 (11.75 g, 56.04 mmol) was added at 0 °C to a solution of D-glucosamine

hydrochloride (10.07 g, 46.7 mmol), K2CO3 (17.43 g, 126.1 mmol) and CuSO4 pentahydrate

(140 mg, 0.467 mmol) in MeOH (234 ml) and the mixture was stirred at room temperature for 3

h. The solution was concentrated and co-evaporated with toluene (2x100 ml). Acetic anhydride

(35.6 ml, 373.6 mmol) and a catalytic amount of DMAP were added to the residue dissolved in

pyridine (120 ml) and the mixture was stirred for 3 h, then concentrated, diluted with water (500

ml) and extracted three times with CH2Cl2 (3x500 ml). The combined organic layers were dried

(Na2SO4), filtered and concentrated. Column flash chromatography [hexane (H)/ethyl acetate

(EA) 6/4] gave the azide 7 (13.41 g, 77%). The spectroscopic characterization data of compound

7 were in agreement with those previously reported.22

Thexyldimethylsilyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-β-D-glucopyranoside (8). A freshly

prepared 2 M solution of hydrazine acetate [NH2NH2·H2O (1.5 ml, 29.20 mmmol), AcOH (1.4

ml, 22.46 mmol) and dry MeOH (15.6 ml)] was added dropwise to a solution of 7 (8.38 g, 22.46

mmol) in dry DMF (100 ml) at 0°C. The mixture was stirred at rt for 2 hours. The mixture was

concentrated, diluted with EA (300 ml) and extracted with water (3 × 300 ml). The organic layer

was dried (Na2SO4), filtered and concentrated to dryness. The obtained residue was dissolved in

dry DMF (100 ml) and cooled to 0°C, then imidazole (4.19 g, 60.6 mmol) and TDSCl (5.94 ml,

30.31 mmol) were added. The mixture was stirred for 24 hours at 0°C. The reaction was

concentrated to reduce the DMF amount, then diluted with EA (150 ml) and washed with water

(2 × 200 mL) and brine (200 mL). The organic phase was dried over Na2SO4 and the solvent

removed under reduced pressure. Flash chromatography (H/EA 7/3) gave 8 as a yellow solid



(7.02 g, 66% from 7). The spectroscopic characterization data of compound 8 were in agreement

with those previously reported.23

Thexyldimethylsilyl 2-azido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-β-D-glucopyranoside

(4). Compound 8 (6.11 g, 12.9 mmol) was treated with a 0.1 M solution of NaOMe in dry MeOH

and stirred at rt under nitrogen atmosphere. Deacetylation was confirmed by TLC (H/EA 7/3),

then the reaction mixture was neutralized by ion-exchange resin (Amberlite IR120, H+ form),

filtered and concentrated. Complete O-deacetylation was further confirmed by 1H-NMR

spectrum of the crude residue. This compound (4.48 g, 12.9 mmol) was suspended in dry

CH3CN (130 ml) and benzaldehyde dimethylacetal (5.8 ml, 38.7 mmol) was added, followed by

addition of a catalytic amount of camphorsulfonic acid. The reaction mixture was vigorously

stirred at rt for 72 h, then it was quenched by addition of Et3N until neutral pH and solvents were

removed under vacuum. The crude was purified by flash chromatography using the Biotage

system (H/EA gradient) to yield 3.56 g of the 4,6-O-benzylidene intermediate. This compound

(3.56 g, 8.17 mmol) was dissolved in dry DMF (70 ml), then BnBr (1.9 ml, 16.2 mmol) and a

catalytic amount of TBAI (0.75 g) were added. 95% NaH (293 mg, 12.2 mmol) was slowly

added and the reaction mixture was stirred at rt for 1 h. The reaction was quenched by MeOH

addition, then the solvents were removed under vacuum. The residue was dissolved in EA (60

ml) and washed with 5% aq. solution of HCl (2 × 200 ml) and brine (1 × 200 ml). The organic

phase was dried over Na2SO4, filtered and concentrated under reduced pressure. The crude

residue was purified by flash chromatography using the Biotage system (H/EA gradient)

affording 4 (3.2 g, 45% yield over three steps) as a white solid. The spectroscopic

characterization data of compound 4 were in agreement with those previously reported.24

Thexyldimethylsilyl 4-O-acetyl-2-azido-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranoside (5).

Compound 4 (4.07 g, 7.74 mmol) was dissolved in dry CH2Cl2 (35 ml) and Et3SiH (6.15 ml, 38.7

mmol) was added dropwise. After 30 min, the mixture is cooled to 0 °C, and trifluoroacetic acid

(2.87 ml, 38.7 mmol) was slowly added. The reaction mixture was stirred at rt and, after

completion (TLC H/EA 95/5), diluted with CH2Cl2, quenched by addition of a satd aq. solution

of NaHCO3. The aqueous phase was extracted with CH2Cl2, the combined organic phases were

dried over Na2SO4, filtered and the solvent removed under reduced pressure. The crude product

was purified by column flash chromatography (H/EA 9/1) to give the alcohol intermediate as a

white solid (3.49 g, 6.61 mmol, 85% yield), whose NMR data were in agreement with those

previously reported.25

The alcohol (2.73 g, 5.18 mmol) was dissolved in dry pyridine (50 ml) and the solution was

cooled to 0°C. Acetic anhydride (987 µl, 10.36 mmol) was slowly added, followed by a catalytic

amount of DMAP. The mixture was stirred at rt until completion (TLC H/EA 85/15). Then, the

reaction mixture was concentrated under reduced pressure and the obtained residue was diluted

with EA (100 ml), washed with aq. 5% solution of HCl (1 × 100 ml), aq. satd NaHCO3 (1 × 100

ml) and brine (1 × 100 ml). The first aqueous phase was extracted with CH2Cl2 (1 × 100 ml) and

the combined organic phases were dried over Na2SO4, filtered and concentrated under reduced

pressure. The crude product was purified by flash chromatography using the Biotage system



(H/EA gradient) affording 5 as a white amorphous solid (2.70 g, 92% yield). [α]D -29.0 (c 1.0,

CHCl3). 1H NMR (400 MHz, CDCl3): δH 7.38–7.24 (m, 10H, Ar), 5.03–4.91 (m, 1H, H-4), 4.81

(d, J 11.5 Hz, 1H, ½ CH2Ph), 4.62 (d, J 11.5 Hz, 1H, ½ CH2Ph), 4.53 (d, J1,2 8.4 Hz, 1H, H-1),

4.51 (s, 2H, CH2Ph), 3.57–3.47 (m, 3H, H-6, H-6’, H-5), 3.41–3.34 (m, 2H, H-3, H-2), 1.85 (s,

3H, CH3CO), 1.68 (hept, J 7.0 Hz, 1H, CH TDS), 0.92 (s, 3H, CH3 TDS), 0.90 (s, 9H, CH3

TDS), 0.22 (s, 3H, CH3Si TDS), 0.21 (s, 3H, CH3Si TDS). 13C NMR (100.6 MHz, CDCl3): δC

169.8 (CO), 97.1 (C-1), 80.5 (C-3), 74.9 (CH2Ph), 73.8 (CH2Ph), 73.6 (C-5), 71.2 (C-4), 70.1 (C-

6), 68.7 (C-2), 34.1 (CH TDS), 25.0 (Cq TDS), 20.9 (CH3CO), 20.1, 20.0, 18.7, 18.6 (4 CH3

TDS), -1.89, -3.09 (2 CH3Si TDS). HRMS (ES) m/z calcd for C30H43N3O6SiNa 592.2819; Found

592.28077 [M+Na]+.

General procedure A. Desilylation. An equimolar amount of glacial acetic acid and a 1 M

solution of tetrabutylammonium fluoride in THF (1.5 mmol of TBAF per silylglycoside mmol)

were sequentially added to a solution of the silylglycoside in anhydrous THF at -40°C. After

completion of the reaction (TLC), the mixture was warmed up to r.t., poured in CH2Cl2, and

washed three times with brine. The organic layer was dried (Na2SO4), filtered and concentrated.

The residue was purified by column chromatography.

2-Azido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-α/β-D-glucopyranose (9). Compound 4 (5.47

g, 9.97 mmol) was treated as described in the General Procedure A. Flash chromatography

(H/EA 7/3) gave hemiacetal 9 as an oil (3.06 g, 80% yield). The spectroscopic characterization

data of compound 9 were in agreement with those previously reported.14,26

4-O-Acetyl-2-azido-3,6-di-O-benzyl-2-deoxy-α/β-D-glucopyranose (11). Compound 5 (2.70 g,

4.74 mmol) was treated as described in the General Procedure A. Flash chromatography (H/EA

7/3) gave hemiacetal 11 as a colourless oil (1.90 g, 94% yield). 1H NMR (400 MHz, CDCl3,

selected NMR signals of the anomeric mixture): δH 7.38–7.24 (m, 10H, Ar), 5.31 (br t, J1,2 J1,OH

2.9 Hz, 1H, H-1 α), 5.01 (dd, J4,3 10.1, J4,5 9.3 Hz, 1H, H-4 α), 4.95 (m, 1H, H-4 β), 4.83 (d, J

11.1 Hz, 1H, ½ CH2Ph α), 4.81 (d, J 11.4 Hz, 1H, ½ CH2Ph β), 4.64 (d, J 11.1 Hz, 1H, ½ CH2Ph

α), 4.61 (d, J 11.4 Hz, 1H, ½ CH2Ph β), 4.60–4.55 (br d, 1H, H-1 β), 4.51 (s, 4H, CH2Ph β,

CH2Ph α), 4.19–4.10 (m, 1H, H-5 α), 4.04–3.92 (m, 1H, H-3 α), 3.60–3.37 (m, 8H, H-5 β, 2 H-6

β, 2 H-6 α, H-2 β, H-2 α, H-3 β), 1.87 (s, 3H, CH3CO α), 1.84 (s, 3H, CH3CO β). 13C NMR

(100.6 MHz, CDCl3, selected NMR signals of the anomeric mixture): δC 169.8 (2 CO α + β),

96.3 (C-1 β), 92.1 (C-1 α), 80.5 (C-3 β), 77.8 (C-3 α), 75.0, 73.7 (CH2Ph β, CH2Ph α), 73.7 (C-5

β), 71.3 (C-4 α), 70.8 (C-4 β), 69.3 (C-5 α), 69.3 (C-6 β, C-6 α), 67.2 (C-2 β), 63.7 (C-2 α), 20.9

(CH3CO β, CH3CO α). MS (ES) m/z calcd for C22H25N3O6Na 450.16; Found 450.5 [M+Na]+.

General procedure B. Synthesis of α-hydrogenphosphonate. A 2 M solution of H3PO3 in

pyridine (3:1 molar ratio as to the hemiacetal) was added dropwise to a solution of the

hemiacetal in dry pyridine (4 ml/mmol), and thereafter the solution was cooled to 0°C and

salicylchlorophosphite (1.5:1 molar ratio as to the hemiacetal) was slowly added. The reaction

was stirred at rt and, after disappearance of the starting material (TLC), the reaction temperature

was raised up to +40°C. The reaction mixture was stirred under argon until complete



disappearance of the β anomer (evidenced by 1H- and 31P-NMR spectra). A 1 M solution of

TEAB (4 ml/mmol) was added to the reaction mixture at rt, then it was diluted with CH2Cl2,

washed three times with cold TEAB solution (0.5 M), dried (Na2SO4), filtered and concentrated.

The crude product was purified by flash chromatography (CH2Cl2/MeOH 8/2 + 1% Et3N). The

obtained compound was further washed with cold TEAB solution (0.25M) to afford the α-H-

phosphonate as a triethylammonium salt.

2-Azido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-α-D-glucopyranosyl hydrogenphosphonate

triethylammonium salt (10). Compound 9 (168 mg, 0.44 mmol) was treated as described in the

General Procedure B, providing α-H-phosphonate 10 (98 mg, 41% yield). [α]D +10.4 (c 1.0,

CHCl3). 1H NMR (400 MHz, CDCl3): δH 7.55-7.22 (m, 10H, Ar), 7.01 (d, JH,P 640.4 Hz, 1H, H-

P), 5.70 (dd, J1,P 8.8, J1,2 3.5 Hz, 1H, H-1), 5.55 (s, 1H, CHPh), 4.92 (d, J 11.1 Hz, 1H, ½

CH2Ph), 4.76 (d, J 11.1 Hz, 1H, ½ CH2Ph), 4.25 (dd, J6,6’ 9.8, J6,5 4.9 Hz, 1H, H-6), 4.21–4.14

(m, 1H, H-5), 4.11 (t, J3,2J3,4 9.8 Hz, 1H, H-3), 3.74-3.68 (m, 2H, H-4, H-6’), 3.47 (ddd, 3J2,P 1.3

Hz, 1H, H-2). 13C NMR (100.6 MHz, CDCl3): δC 101.6 (CHPh), 93.8 (C-1), 82.9 (C-4), 76.3 (C-

3), 74.9 (CH2Ph), 69.0 (C-6), 63.6 (C-2), 63.5 (C-5). 31P NMR (162 MHz, CDCl3): δP 1.84. MS

(ES) m/z calcd for C20H21N3O7P 446.11; Found 446.3 [M]-.

4-O-Acetyl-2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl hydrogenphosphonate

triethylammonium salt (12). Compound 11 (368 mg, 0.86 mmol) was treated as described in

the General Procedure B, providing α-H-phosphonate 12 (265 mg, 52% yield). [α]D +9.7 (c 1.1,

CHCl3). 1H NMR (400 MHz, CDCl3): δH 7.38–7.19 (m, 10H, Ar), 7.03 (d, JH,P 642.0 Hz, 1H, H-

P), 5.73 (dd, J1,P 8.7, J1,2 3.0 Hz, 1H, H-1), 5.14 (t, J4,3 J4,5 9.7 Hz, 1H, H-4), 4.79 (d, J 11.1 Hz,

1H, ½ CH2Ph), 4.60 (d, J 11.1 Hz, 1H, ½ CH2Ph), 4.49 (d, J 11.8 Hz, 1H, ½ CH2Ph), 4.44 (d, J

11.8 Hz, 1H, ½ CH2Ph), 4.19 (m, 1H, H-5), 4.05 (t, J3,2 9.7 Hz, 1H, H-3), 3.57–3.38 (m, 3H, H-

6, H-6’, H-2), 1.83 (s, 3H, CH3CO). 13C NMR (100.6 MHz, CDCl3): δC 169.7 (CH3CO), 93.1

(C-1), 78.0 (C-3), 74.8 (CH2Ph), 73.6 (CH2Ph), 71.0 (C-4), 70.2 (C-5), 69.0 (C-6), 63.6 (C-2),

20.8 (CH3CO). 31P NMR (162 MHz, CDCl3): δP 2.24. MS (ES) m/z calcd for C22H25N3O8P

490.14; Found 490.3 [M]-.

General procedure C. Hydrogenphosphonate coupling and oxidation. The alcohol and the

H-phosphonate (10 or 12) were co-evaporated with dry pyridine for three times under high

vacuum. The residue was then dissolved in dry pyridine(10 ml/mmol), and PivCl (2.5:1 molar

ratio as to the H-phosphonate) was added. The mixture was stirred under nitrogen atmosphere for

45 min-1 h (TLC in CH2Cl2/MeOH mixtures). After cooling to -40°C, a freshly prepared 0.5 M

solution of I2 (2.5:1 molar ratio as to the H-phosphonate) in a 19:1 mixture of pyridine-H2O was

added. The oxidation was completed at 0°C and quenched by dropwise addition of a 0.5 M

solution of Na2S2O3·5H2O (10% w/v). The reaction mixture was diluted with CHCl3, and the

organic layer was washed two times with a 0.5 M solution of Na2S2O3·5H2O (10% w/v), then 0.5

M TEAB, dried (Na2SO4), filtered, and concentrated. The crude residue was purified by flash

chromatography (CH2Cl2/MeOH + 1% of Et3N), providing the phosphodiester derivative. The

obtained compound was further washed with cold TEAB solution (0.25 M) to afford the

corresponding triethylammonium salt.



General procedure D. Azide reduction and N-acetylation. To a solution of the azide-

containing compound in MeOH (10 ml/mmol), first NiCl2·6H2O (3:1 molar ratio as to the

number of azido groups) was added under stirring and N2 atmosphere, then NaBH4 (8:1 molar

ratio as to the number of azido groups) portionwise (1 hour) at 0 °C. A black precipitate

indicated the formation of a Ni-B species. The mixture was stirred at 0 °C and, after consumption

of the starting material (amine formation is monitored by ninhydrin-detection), Ac2O (20:1 molar

ratio as to the azide-containing compound) was added. The mixture was concentrated under

reduced pressure, diluted with CH2Cl2 and washed three times with water. The organic phase

was dried (Na2SO4), filtered and concentrated, and purified by flash chromatography to give the

acetamide. The obtained compound was further washed with cold TEAB solution (0.25 M) to

afford the corresponding triethylammonium salt.

General procedure E: Hydrogenolysis and ion-exchange. The protected acetamide was

hydrogenolysed over Pd/C (10%) in a 5:1 mixture of MeOH and H2O at room temperature for

24-48 h. The mixture was filtered over a Celite pad and the filtrate was concentrated. Then the

residue was dissolved in H2O and first eluted through a column filled with Dowex 50W-X8 resin

(H+ form), and then through a column filled with the same resin in Na+ form. The eluate was

concentrated and lyophilized to afford the target compound as sodium salt.

3-(N-Carbobenzyloxy)aminopropyl 1-O-(2-azido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-α-

D-glucopyranosyl phosphate) triethylammonium salt (13). Benzyl N-(3-

hydroxypropyl)carbamate (136 mg, 0.65 mmol) was condensed with H-phosphonate 10 (145 mg,

0.26 mmol) in pyridine (1.3 ml) in the presence of PivCl (0.04 ml, 0.32 mmol), and in situ

oxidized with I2 in pyridine-H2O (2 ml) according to General Procedure C. Flash

chromatography (CH2Cl2/MeOH 8/2 + 1% of Et3N) and subsequent TEAB washing afforded

glycosyl phosphodiester 13 as a colourless oil (122 mg, 62% yield). [α]D +11.1 (c 1.0, CHCl3). 1H NMR (400 MHz, CDCl3): δH 7.50–7.43 (m, 2H, Ar), 7.42–7.23 (m, 13H, Ar), 6.18 (br t, J 5.3

Hz, 1H, NHCbz), 5.65 (dd, JH,P 7.5, J1,2 3.5 Hz, 1H, H-1), 5.55 (s, 1H, CHPh), 5.06 (s, 2H,

CH2Ph), 4.93 (d, J 11.1 Hz, 1H, ½ CH2Ph), 4.76 (d, J 11.1 Hz, 1H, ½ CH2Ph), 4.24 (dd, J6,6’

10.1, J6,5 4.9 Hz, 1H, H-6), 4.20–3.94 (m, 4H, H-5, H-3, OCH2CH2CH2N), 3.743.66 (m, 2H, H-

4, H-6’), 3.47 (ddd, J2,3 9.8, J2,P 2.3 Hz, 1H, H-2), 3.35 (dd, J 11.7, 5.9 Hz, 2H,

OCH2CH2CH2N), 1.83–1.72 (m, 2H, OCH2CH2CH2N). 13C NMR (100.6 MHz, CDCl3): δC 156.7

(CO), 101.6 (CHPh), 94.6 (C-1), 82.8 (C-4), 76.3 (C-3), 74.9 (C-6), 69.1 (CH2Ph), 66.5 (CH2Ph),

63.7 (C-2), 63.6 (C-5), 63.04 (OCH2CH2CH2N), 37.46 (OCH2CH2CH2N), 30.45

(OCH2CH2CH2N). 31P NMR (162 MHz, CDCl3): δP -0.84. HRMS (ES) m/z calcd for

C31H34N4O10P 653.2018; Found 653.20169 [M]-.

3-(N-Carbobenzyloxy)aminopropyl 1-O-(2-acetamido-3-O-benzyl-4,6-O-benzylidene-2-

deoxy-α-D-glucopyranosyl phosphate), triethylammonium salt (14). Compound 13 (97 mg,

0.13 mmol) was converted into acetamide 14 as described in the General Procedure D. Flash

chromatography (EA/MeOH from 9/1 to 8/2 + 1% of Et3N) and subsequent TEAB washing

afforded glycosyl phosphodiester 14 (29 mg, 29% yield). [α]D +31.2 (c 0.4, MeOH). 1H NMR

(400 MHz, CDCl3): δH 7.53–7.44 (m, 2H, Ar), 7.44–7.20 (m, 13H, Ar), 6.76 (d, J 9.9 Hz, 1H,



NHAc), 5.99 (t, J 5.5 Hz, 1H, NHCbz), 5.56 (s, 1H, CHPh), 5.47 (dd, J1,P 7.2, J1,2 3.5 Hz, 1H, H-

1), 5.05 (s, 2H, CH2Ph), 4.88 (d, J 12.1 Hz, 1H, ½ CH2Ph), 4.66 (d, J 12.1 Hz, 1H, ½ CH2Ph),

4.36–4.27 (m, 1H, H-2), 4.21 (dd, J6,6’ 10.2, J6,5 4.9 Hz, 1H, H-6), 4.10–4.02 (m, 1H, H-4), 4.02–

3.87 (m, 2H, OCH2CH2CH2N), 3.86–3.68 (m, 3H, H-3, H-5, H-6’), 3.32 (dd, J 12.3, 6.2 Hz, 2H,

OCH2CH2CH2N), 1.91 (s, 3H, NHCOCH3), 1.81–1.71 (m, 2H, OCH2CH2CH2N). 13C NMR

(100.6 MHz, CDCl3): δC 156.7 (CO), 101.6 (CHPh), 94.6 (C-1), 82.8 (C-4), 76.3 (C-3), 74.9 (C-

6), 69.1 (CH2Ph), 66.5 (CH2Ph), 63.7 (C-2), 63.6 (C-5), 63.0 (OCH2CH2CH2N), 37.46

(OCH2CH2CH2N), 30.45 (OCH2CH2CH2N). 31P NMR (162 MHz, CDCl3): δP -0.49. MS (ES) m/z

calcd for C33H38N2O11P 669.22; Found 669.4 [M]-.

3-Aminopropyl 1-O-(2-acetamido-2-deoxy-α-D-glucopyranosyl phosphate), sodium salt (1).

The acetamide 14 (26 mg, 0.034 mmol) was hydrogenolysed over Pd/C in MeOH:H2O (4 mL)

and subjected to ion exchange according to General Procedure E, affording monomer 1 as a foam

(12 mg, 93% yield). [α]D +66.0 (c 0.1, H2O). 1H NMR (400 MHz, D2O): δH 5.43 (dd, J1,P 7.4, J1,2

3.3 Hz, 1H, H-1), 4.03–3.73 (m, 7H, OCH2CH2CH2N, H-2, H-6, H-6’, H-3, H-5), 3.51 (t, J4,3 J4,5

9.3 Hz, 1H, H-4), 3.20–3.04 (m, 2H, OCH2CH2CH2N), 2.08–2.01 (m, 3H, NHCOCH3), 2.01–

1.87 (m, 2H, OCH2CH2CH2N). 13C NMR (100.6 MHz, D2O): δC 94.2 (C-1), 72.9 (C-3), 70.1 (C-

5), 69.4 (C-4), 63.3 (OCH2CH2CH2N), 60.2 (C-6), 53.7 (C-2), 36.9 (OCH2CH2CH2N), 27.6

(OCH2CH2CH2N), 21.7 (NHCOCH3). 31P NMR (162 MHz, D2O): δP -0.94. HRMS (ES) m/z

calcd for C11H22N2O9P 357.1068; Found 357.10632 [M]-; calcd for C11H23N2O9PNa 381.1044;

Found 381.10368 [M+H+Na]+; calcd for C11H23N2O9PNa2 404.0942; Found 404.08561

[M+H+2Na]2+.

3-(N-Carbobenzyloxy)aminopropyl 1-O-(4-O-acetyl-2-azido-3,6-di-O-benzyl-2-deoxy-α-D-

glucopyranosyl phosphate), triethylammonium salt (15). Benzyl N-(3-

hydroxypropyl)carbamate (136 mg, 0.65 mmol) was condensed with H-phosphonate 12 (76 mg,

0.13 mmol) in pyridine (1.3 ml) in the presence of PivCl (0.04 ml, 0.32 mmol), and in situ

oxidized with I2 in pyridine-H2O (2 ml) according to General Procedure C. Flash

chromatography (CH2Cl2/MeOH from 9/1 to 8/2 + 1% of Et3N) and subsequent TEAB washing

afforded glycosyl phosphodiester 15 as a colourless oil (66 mg, 64% yield). [α]D +35.8 (c 1.2,

CHCl3). 1H NMR (400 MHz, CDCl3): δH 7.33–7.17 (m, 15H, Ar), 6.20 (t, J 5.5 Hz, 1H, NH),

5.62 (dd, J1,P =7.8, J1,2 3.1 Hz, 1H, H-1), 5.06 (t, J4,3 J4,5 9.7 Hz, 1H, H-4), 5.02 (s, 2H, CH2Ph),

4.74 (d, J 11.1 Hz, 1H, ½ CH2Ph), 4.56 (d, J 11.1 Hz, 1H, ½ CH2Ph), 4.51 (d, J 11.8 Hz, 1H, ½

CH2Ph), 4.48 (d, J 11.8 Hz, 1H, ½ CH2Ph) 4.20–4.09 (m, 1H, H-5), 4.05–3.90 (m, 3H, H-3,

OCH2CH2CH2N), 3.56–3.46 (m, 3H, H-2, H-6, H-6’), 3.27 (dd, J 11.5, 5.7 Hz, 2H,

OCH2CH2CH2N), 1.81 (s, 3H, CH3CO), 1.73–1.55 (m, 2H, OCH2CH2CH2N). 13C NMR (100.6

MHz, CDCl3): δC 169.7, 156.6 (CO), 93.7 (C-1), 77.7 (C-3), 74.7 (CH2Ph), 73.5 (CH2Ph), 70.9

(C-4), 69.9 (C-5), 69.0 (C-6), 66.3 (CH2Ph), 63.7 (C-2), 62.8 (OCH2CH2CH2N), 37.28

(OCH2CH2CH2N), 30.2 (OCH2CH2CH2N), 20.79 (CH3CO). 31P NMR (162 MHz, CDCl3): δP -

0.52. HRMS (ES) m/z calcd for C33H38N4O11P 697.2280; Found 697.22780 [M]-.

3-(N-Carbobenzyloxy)aminopropyl 1-O-[2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyran-

osyl phosphate 4-(2-azido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-α-D-glucopyranosyl phos-



phate)], triethylammonium salt (17). A solution of 15 (120 mg, 0.15 mmol) in dry MeOH was

treated with 0.05 M NaOMe in MeOH and stirred under nitrogen atmosphere. After reaction

completion, the mixture was diluted with MeOH, neutralized with Amberlite IR 120 (H+) resin,

filtered, and concentrated. The crude was purified by flash chromatography (CH2Cl2/MeOH 8/2

+ 1% of Et3N), providing the corresponding deacetylated product 16 (110 mg), which was

condensed with H-phosphonate 10 (66 mg, 0.12 mmol) in pyridine (1.5 ml) in the presence of

PivCl (0.045 ml, 0.36 mmol), and in situ oxidized with I2 in pyridine-H2O (2 ml) according to

General Procedure C. Flash chromatography (CH2Cl2/MeOH 8/2 + 1% of Et3N) and subsequent

TEAB washing afforded glycosyl phosphodiester 17 (71 mg, 45% yield). [α]D +12.7 (c 1.15,

CHCl3). 1H NMR (400 MHz, CDCl3): δH 7.58–7.41 (m, 5H, Ar), 7.41–7.13 (m, 20H, Ar), 6.34 (t,

J 5.9 Hz, 1H, NHCbz), 5.78–5.67 (m, 2H, H-1a, H-1b), 5.56 (s, 1H, CHPh), 5.34 (d, J 11.0 Hz,

1H, ½ CH2Ph), 5.06 (s, 2H, CH2Ph), 4.82 (d, 1H, ½ CH2Ph), 4.79 (d, 1H, ½ CH2Ph), 4.67 4.55

(m, 3H, ½ CH2Ph, CH2Ph), 4.42 (t, J4a,3a = J4a,5a 10.0 Hz, 1H, H-4a), 4.34–4.16 (m, 3H, H-6b, H-

5a, H-5b), 4.13–3.93 (m, 5H, H-6a, H-3b, H-3a, OCH2CH2CH2N), 3.87 (dd, J6’a,6a 10.9, J6’a,5a 6.2

Hz, 1H, H-6’a), 3.77–3.65 (m, 2H, H-6’b, H-4b), 3.52–3.35 (m, 2H, H-2a, H-2b), 3.30 (dd, J

10.9, 5.2 Hz, 2H, OCH2CH2CH2N), 1.69–1.52 (m, 2H, OCH2CH2CH2N). 13C NMR (100.6 MHz,

CDCl3): δC 101.5 (CHPh), 94.5 (C-1b), 93.8 (C-1a), 82.8 (C-4b), 79.0 (C-3b), 76.5 (C-3a), 74.8

(CH2Ph), 74.7 (C-4a), 74.4 (CH2Ph), 73.4 (CH2Ph), 72.2 (C-5a), 69.9 (C-6a), 69.0 (C-6b), 66.2

(CH2 Cbz), 64.0 (C-2b), 63.7 (C-2a), 63.3 (C-5b), 62.7 (OCH2CH2CH2N), 37.25

(OCH2CH2CH2N), 30.17 (OCH2CH2CH2N). 31P NMR (162 MHz, CDCl3): δP -0.23, -1.99.

HRMS (ES) m/z calcd for C51H55N7O17P2Na 1122.3033; Found 1122.30177 [M+Na]-; calcd for

C51H55N7O17P2 1099.3135; Found 549.65634 [M]2-.

3-Aminopropyl 1-O-[2-acetamido-2-deoxy-α-D-glucopyranosyl phosphate 4-(2-acetamido-2-

deoxy-α-D-glucopyranosyl phosphate)], bis-sodium salt (2). Compound 17 (95 mg, 0.073

mmol) was converted into acetamide as described in the General Procedure D. Flash

chromatography (CH2Cl2/MeOH from 8/2 to 7/3 + 1% of Et3N) and subsequent TEAB washing

afforded the bis-acetamido glycosyl phosphodisaccharide, which was hydrogenolysed over Pd/C

in MeOH:H2O (4 ml) and subjected to ion exchange according to General Procedure E, affording

dimer 2 as a foam (18 mg, 36% yield over two steps). [α]D +19.0 (c 0.07, H2O). 1H NMR (400

MHz, D2O): δH 5.17 (dd, J1a,P 7.5 Hz, J1a,2a 3.3 Hz, 1H, H-1a), 4.70 (dd, J1b,P 8.3 Hz, J1b,2b 3.8

Hz, 1H, H-1b), 4.12–3.60 (m, 10H, H-2a or H-2b, H-3a, H-3b, H-4a, H-5a, H-5b, H-6a, H-6’a,

OCH2CH2CH2N), 3.54–3.34 (m, 4H, H-2b or H-2a, H-4b, H-6b, H-6’b), 3.16 3.10 (m, 2H,

OCH2CH2CH2N), 2.01 (br s, 6H, 2 × NHCOCH3), 1.97 1.91 (m, 2H, OCH2CH2CH2N). 13C

NMR (100.6 MHz, D2O): δC 94.8, 90.7 (C-1a, C-1b), 75.8, 73.7 (C-3a, C-3b), 71.4, 70.5 (C-5a,

C-5b), 70.4, 70.0 (C-4a, C-4b), 68.3 (OCH2CH2CH2N), 60.6, 60.4 (C-6a, C-6b), 56.5, 53.9 (C-

2a, C-2b), 22.0 (NHCOCH3), 21.7 (OCH2CH2CH2N), 19.9 (OCH2CH2CH2N). 31P NMR (162

MHz, D2O): δP 0.90, 0.60. HRMS (ES) m/z calcd for C19H34N3O17P2Na 661.1267; Found

661.77595 [M+Na]-.

3-(N-Carbobenzyloxy)aminopropyl 1-O-[2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyran-

osyl phosphate 4-(4-O-acetyl-2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl phos-



phate)], bis-triethylammonium salt (18). Alcohol 16 (425 mg, 0.56 mmol) was condensed with

H-phosphonate 12 (366 mg, 0.62 mmol) in pyridine (2.5 ml) in the presence of PivCl (0.14 ml,

1.11 mmol), and in situ oxidized with I2 in pyridine-H2O (2 ml) according to General Procedure

C. Flash chromatography (CH2Cl2/MeOH 85/15 + 1% of Et3N) and subsequent TEAB washing

afforded glycosyl phosphodiester 18 (290 mg, 40% yield). The identity of compound 18 was

confirmed by 1H- and 31P-NMR, and it was used in the following steps without further

characterization. 1H NMR (400 MHz, CDCl3): δH 7.53–7.15 (m, 25H, Ar), 6.29 (br t, J 5.8 Hz,

1H, NHCbz), 5.72 (dd, J1,P 7.5 Hz, J1,2 3.3 Hz, 1H, H-1a or H-1b), 5.67 (dd, J1,P 8.2 Hz, J1,2 3.4

Hz, 1H, H-1b or H-1a), 5.28 (d, J 11.0 Hz, 1H, ½ CH2Ph), 1H), 5.14 (t, J4b,3b = J4b,5b 9.7 Hz, 1H,

H-4b), 5.02 (br s, 2H, CH2Ph), 4.75 (d, J 11.0 Hz, 1H, ½ CH2Ph), 4.63 (d, J 11.5 Hz, 1H, ½

CH2Ph), 4.58 (br s, 2H, CH2Ph), 4.47–4.36 (m, 4H, H-4a, ½ CH2Ph, CH2Ph), 4.24 (br t, 1H, H-

5a or H-5b), 4.17 (dt, J5,4 10.2 Hz, J5,6 3.5 Hz, 1H, H-5b or H-5a), 4.11–3.89 (m, 5H, H-3a, H-3b,

H-6a, OCH2CH2CH2N), 3.85 (dd, J6’a,6a 11.1 Hz, J6’a,5a 6.5 Hz, 1H, H-6’a), 3.49–3.37 (m, 4H, H-

2a, H-2b, H-6b, H-6’b), 3.25 (dd, J 11.8, 6.1 Hz, 2H, OCH2CH2CH2N), 1.75 (s, 3H, CH3CO),

1.61–1.52 (m, 2H, OCH2CH2CH2N).). 31P NMR (162 MHz, CDCl3): δP -0.90, -2.77.

3-(N-Carbobenzyloxy)aminopropyl 1-O-{2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyran-

osyl phosphate 4-[2-azido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-α-D-glucopyranosyl phos-

phate 4-(2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl phosphate)]}, tris-triethyl-

ammonium salt (20). A solution of 18 (290 mg, 0.22 mmol) in dry MeOH was treated with 0.05

M NaOMe in MeOH and stirred under nitrogen atmosphere. After reaction completion, the

mixture was diluted with MeOH, neutralized with Amberlite IR 120 (H+) resin, filtered, and

concentrated. The crude was purified by flash chromatography (CH2Cl2/MeOH 85/15 + 1% of

Et3N), providing the corresponding deacetylated product 19 (228 mg, 0.16 mmol), which was

condensed with H-phosphonate 10 (142 mg, 0.24 mmol) in pyridine (2.5 ml) in the presence of

PivCl (0.074 ml, 0.6 mmol), and in situ oxidized with I2 in pyridine-H2O (2 ml) according to

General Procedure C. Flash chromatography (CH2Cl2/MeOH 8/2 + 1% of Et3N) and subsequent

TEAB washing afforded glycosyl phosphodiester 20 as an amorphous solid (190 mg, 43% yield

from 18). [α]D +12.8 (c 1.0, CHCl3). 1H NMR (400 MHz, CDCl3): δH 7.59–7.04 (m, 35H, Ar),

6.32 (s, 1H, NHCbz), 5.78 (dd, J1a,P 7.0, J1a,2a =2.4 Hz, 1H, H-1a), 5.74–5.58 (m, 2H, H-1b, H-

1c), 5.52 (s, 1H, CHPh), 5.41–5.16 (m, 2H, 2 × ½ CH2Ph), 5.03 (s, 2H, CH2Ph), 4.83–4.70 (m,

2H, 2 × ½ CH2Ph), 4.68–4.52 (m, 5H, 3 × ½ CH2Ph, CH2Ph), 4.52–4.32 (m, 2H, H-4b, H-4a),

4.32–3.76 (m, 12H, H-6c, H-5a, H-5b, H-6a, H-3b, H-3a, H-6b, H-3c, OCH2CH2CH2N, H-6’a,

H-6’b), 3.76–3.53 (m, 2H, H-6’c, H-4c), 3.47 3.28 (m, 3H, H-2a, H-2b, H-2c), 3.25 (d, J 5.2

Hz, 2H, OCH2CH2CH2N), 1.62–1.45 (m, 2H, OCH2CH2CH2N). 13C NMR (100.6 MHz, CDCl3):

δC 101.56 (CHPh), 94.6 (C-1c), 94.1, 93.9 (C-1a, C-1b), 82.9 (C-4c), 79.5, 79.0 (C-3a, C-3b),

76.7 (C-3c), 75.1 (C-4a), 74.9 (CH2Ph), 74.7 (C-4b), 74.5 (CH2Ph), 73.5, 73.4 (CH2Ph), 72.3 (C-

5a), 72.3 (C-5b), 70.0 (C-6b), 69.7 (C-6a), 69.0 (C-6c), 66.3 (CH2Ph), 64.1, 64.0 (C-2a, C-2b),

63.8 (C-2c), 63.3 (C-5c), 62.8 (OCH2CH2CH2N), 37.3 (OCH2CH2CH2N), 29.8

(OCH2CH2CH2N). 31P NMR (162 MHz, CDCl3): δP -0.30, -2.26 (2 P). HRMS (ES) m/z calcd for

C71H77N10O24P3 1546.4336; Found 773.21635 [M+H]2-; calcd for C71H76N10O24P3Na 1568.4150;


3 ©ARKAT-USA, Inc.

Found 784.20616 [M+Na]2-; calcd for C71H76N10O24P3Na2 1591.4048; Found 1591.39910

[M+2Na]-.

3-Aminopropyl 1-O-[2-acetamido-2-deoxy-α-D-glucopyranosyl phosphate 4-[2-acetamido-2-

deoxy-α-D-glucopyranosyl phosphate 4-(2-acetamido-2-deoxy-α-D-glucopyranosyl

phosphate)]], tris-sodium salt (3). Compound 20 (80 mg, 0.043 mmol) was converted into

acetamide as described in the General Procedure D. Flash chromatography (CH2Cl2/MeOH 7/3 +

1% of Et3N) and subsequent TEAB washing afforded the tris-acetamido glycosyl

phosphotrisaccharide, which was hydrogenolysed over Pd/C in MeOH:H2O (4 ml) and subjected

to ion exchange according to General Procedure E, affording trimer 3 as a foam (14 mg, 33%

yield over two steps). [α]D +86.6 (c 0.4, H2O). 1H NMR (400 MHz, D2O): δH 5.55–5.45 (m, 2H,

H-1a, H-1b), 5.45–5.35 (m, 1H, H-1c), 4.16–3.61 (m, 19H, OCH2CH2CH2N, H-2a, H-2b, H-2c,

H-6a, H-6b, H-6c, H-6’a, H-6’b, H-6’c, H-3a, H-3b, H-3c, H-5a, H-5b, H-5c, H-4a, H-4b), 3.51

(t, J4c,3c = J4c,5c 9.5 Hz, 1H, H-4c), 3.10 (t, J 7.0 Hz, 1H, OCH2CH2CH2N), 2.07–1.88 (m, 11H, 3

× NHCOCH3, OCH2CH2CH2N). 13C NMR (100.6 MHz, D2O): δC 174.6 (2 CO),175.8 (CO),

94.5 (C-1b), 94.2 (C-1c), 93.6 (C-1a), 74.0 (C-3a, C-3b), 73.1 (C-3c), 72.2 (C-5a, C-5b), 70.9

(C-5c), 70.1, 69.6 (C-4a, C-4b, C-4c), 63.4 (OCH2CH2CH2N), 60.3 (C-6a, C-6b, C-6c), 53.7 (C-

2a, C-2b, C-2c), 37.2 (OCH2CH2CH2N), 27.7 (OCH2CH2CH2N), 22.2 (3 × NHCOCH3). 31P

NMR (162 MHz, D2O): δP -0.31, -0.65, -0.71. HRMS (ES) m/z calcd for C27H48N4O25P3Na2

967.1632; Found 967.16133 [M+2Na]-; calcd for C27H49N4O25P3Na 945.1707; Found 945.17800

[M+H+Na]-; calcd for C27H48N4O25P3Na 944.1729; Found 472.08681 [M+Na]2-; calcd for

C27H49N4O25P3 922.1904; Found 461.09538 [M+H]2-.

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synthesis and conjugation of neisseria meningitidis x

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